BirA, also known as biotin protein ligase (BPL), is a multifunctional monomeric enzyme that belongs to the group II biotin-protein ligase family. It is also referred to as biotin operon repressor, biotin-[acetyl-CoA-carboxylase] ligase, and biotin-[acetyl-CoA-carboxylase] synthetase. In Escherichia coli, BirA serves dual roles: as a DNA-binding protein that represses the biotin biosynthesis operon and as an enzyme catalyzing the ATP-dependent biotinylation of specific lysine residues on biotin-accepting proteins or peptides, such as carboxyl carrier protein (BCCP) and AviTag fusion proteins.

Structurally, BirA comprises an N-terminal helix-turn-helix DNA-binding domain, a catalytic core responsible for synthesizing biotinyl 5'-adenylate (bio-5'-AMP), and a C-terminal domain that contributes to DNA binding, dimerization, and catalytic activity. The enzyme's GRGRRG motif near the biotin-binding site facilitates ATP binding, and its dynamic active-site loop undergoes structural ordering upon biotin binding.

BirA-mediated precise biotinylation enables the use of (strept)avidin-biotin interactions in diverse biochemical and cell biology applications, including protein capture, immobilization, multimerization, and molecular bridging. With its exceptional specificity, BirA biotinylates only one out of over 4,000 proteins in E. coli, ensuring targeted modification critical for research and industrial processes. This versatility makes it a powerful tool in protein engineering, affinity studies, and diagnostic development.